Method for inhibiting binding to B-cell receptor

ABSTRACT

The invention provides an isolated protein which is a member of the TNF ligand superfamily and comprises: i) a polypeptide having the amino acid sequence of figure (1); or ii) a variant of the polypeptide of i).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/745,684, filed May 8, 2007, now patented as U.S. Pat. No. 8,216,576,which is a continuation of U.S. patent application Ser. No. 09/868,533,filed Sep. 21, 2001, now patented as U.S. Pat. No. 7,399,593, which is a371 of International Application No. PCT/EP99/07303, filed Oct. 5, 1999,which claims priority to Great Britain Application No. 9828628.9, filedDec. 23, 1998.

The present invention relates to a novel protein of the TNF ligandsuperfamily, nucleotides coding for it, vectors and host cellscontaining the same and methods of screening for modulators of theinteraction between said protein and its receptor, said modulators foruse in therapy for various disorders including, but not restricted to,cancer, inflammation, infection and autoimmune disease. Also, direct useof said ligand in therapy, for example against viral diseases or as apotential vaccine adjuvant.

Fifteen other members of the TNF ligand family have currently beencloned and published and most have been shown to bind to cell-surfacereceptors of the TNF receptor family. The interaction between a TNFligand and its receptor is the key signal to start a chain of eventsleading to a range of responses as diverse as T-cell proliferation,apoptosis and induction of cytokine production. Some activities such asinduction of T-cell proliferation are common to many members of thefamily, whilst some are shared by only a few, and others are unique. Theinteraction between these ligands and their receptors provides anattractive target for the development of novel therapies.

The present invention provides an isolated protein comprising i) apolypeptide having the amino acid sequence of FIG. 1 or ii) a variant ofthe polypeptide of i). The invention also provides an isolated proteincomprising a polypeptide having the amino acid sequence as provided inFIG. 2 or variants thereof. The protein having the amino acid sequenceprovided in FIG. 2 is obtainable from humans and is a type II membraneprotein with a single transmembrane domain near the N-terminus, whichcontains two potential N-linked glycosylation sites, and a proteasecleavage site between amino acids arginine 133 and alanine 134. Thepolypeptide having the amino acid sequence provided in FIG. 1 issoluble, and forms part of the extracellular region of the polypeptidehaving the amino acid sequence provided in FIG. 2. Preferably theprotein of the invention comprises a polypeptide which is 65%,preferably 75%, more preferably 80% and even more preferably 90%homologous to the amino acid sequence of FIG. 1. The protein of theinvention most preferably comprises a polypeptide which is, at least95%, for example 97%, 98% or 99% homologous to the amino acid sequenceof FIG. 1. Preferably the protein of the invention is obtainable frommammals, more preferably from mice or humans, and most preferably fromhumans.

The present invention further provides a protein comprising apolypeptide which has the sequence as provided in FIG. 2 from amino acid134 onwards, or the sequence as provided in FIG. 6 from amino acid 127onwards.

The present invention further provides a protein comprising apolypeptide which has the sequence as provided in FIG. 2 from amino acid122 onwards.

The present invention further provides a protein comprising apolypeptide which has the sequence as provided in FIG. 6 from amino acid115 onwards.

The present invention further provides an isolated protein comprising apolypeptide having the amino acid sequence as provided in FIG. 5, orvariants thereof. Moreover, the invention provides an isolated proteincomprising a polypeptide having the amino acid sequence as provided inFIG. 6 or variants thereof. The protein having the amino acid sequenceas provided in FIG. 6 is isolatable from mice and is a type II membraneprotein with a single transmembrane domain near the N-terminus, whichprotein contains one potential N-linked glycosylation site, and aprotease cleavage site between amino acids arginine126 and alanine127.The polypeptide having the amino acid sequence provided in FIG. 5 issoluble, and forms part of the extracellular region of the polypeptidehaving the amino acid sequence provided in FIG. 6.

Proteins of the invention isolatable from humans, and proteins of theinvention isolatable from mice are highly homologous, displaying 67%amino acid identity over their entire sequence. In the C-terminal regioninvolved in receptor binding, amino acid identity is much higher (87%).A significant difference between proteins of the invention isolatablefrom humans or mice is the presence of an additional exon in the mousesequence encoding an extra 31 amino acids which reduces the overallhomology between the two proteins.

The term variant refers to proteins which have substantially the samebiological functionality as the protein for which sequence informationhas been provided. The term variant encompasses fragments, derivativesand analogues of the protein of the invention.

Fragments include portions of the protein which retain sufficientidentity to the original protein to be effective for example in ascreen.

Derivatives include alternate forms of the protein sequence which mayhave deletions, additions or substitutions of one or more amino acids.It will be understood by a person skilled in the art that certainsubstitutions, deletions or additions of amino acids can be made, orindeed can occur naturally, without substantially altering the functionof the protein.

Analogues include but are not limited to precursor proteins which can beactivated by cleavage of the precursor protein to produce an activemature protein, or a fusion with a leader or secretory sequence to aidpurification.

The protein of the present invention may be a recombinant protein, anatural protein or a synthetic protein.

The proteins of the invention may be present in all embodiments intrimeric form and such trimers form an embodiment of the invention.Typically the proteins of the invention will bind to their receptor as atrimer, thus allowing two or more receptor molecules to be brought intoproximity. A trimer may be a heterotrimer wherein more than one type ofsubunit is present, or a homotrimer wherein all subunits are the same.

The present invention also provides antibodies specific for the proteinof the invention. The term antibody as used herein includes allimmunoglobulins and fragments thereof which contain recognition sitesfor antigenic determinants of proteins of the present invention. Theantibodies of the present invention may be polyclonal or monoclonal, maybe intact antibody molecules or fragments containing the active bindingregion of the antibody, e.g. Fab or F(ab)₂. The present invention alsoincludes chimeric, single chain and humanised antibodies and fusionswith non-immunoglobulin molecules. Various procedures known in the artmay be used for the production of such antibodies and fragments.

The proteins of the invention, their variants or cells expressing themcan be used as an immunogen to produce antibodies thereto. Antibodiesgenerated against the proteins of the invention can be obtained bydirect injection of the polypeptide into an animal, preferably anon-human. The antibody so obtained will then bind the protein itself.In this manner, even a fragment of the protein of the invention can beused to generate antibodies binding the whole native protein.

The antibodies of the present invention may be used to locate theprotein of the invention in tissue expressing that protein. They arealso, for example, useful for purification of a protein of theinvention, and accordingly there is provided a method of purifying aprotein of the invention which method comprises the use of an antibodyof the present invention. The antibodies of the present invention mayalso be used as therapeutic agents in their own right.

A further aspect of the invention provides an isolated polynucleotidewhich encodes a protein of the invention. Also included within theinvention are anti-sense nucleotides or complementary strands.Preferably the nucleotide encodes a protein of the invention isolatablefrom a mouse or a human. More preferably the isolated polynucleotidecomprises the polynucleotide portion having the nucleotide sequenceshown in FIG. 3 (SEQ ID NO: 3), which codes for the polypeptide shown inFIG. 3 (SEQ ID NO: 9), a variant of said portion, or a complementarystrand. The present invention further provides an isolatedpolynucleotide comprising the nucleotide sequence shown in FIG. 4 (SEQID NO: 4), which codes for the polypeptide of FIG. 4 (SEQ ID NO:10).

The nucleotide sequence may be isolated from a cell (preferably a humancell), by screening with a probe derived from the protein of theinvention, or by other methodologies known in the art such as polymerasechain reaction (PCR) for example on genomic DNA with appropriateoligonucleotide primers derived from or designed based on the protein ofthe invention. A bacterial artificial chromosome library can begenerated using mouse or human DNA for the purposes of screening.

The nucleotide sequences of the present invention may be in the form ofRNA or in the form of DNA, for example cDNA, genomic DNA, and syntheticDNA. Preferably the nucleotide sequence of the invention is cDNA. TheDNA may be double-stranded or single-stranded, and if single strandedmay be the coding strand or non-coding (anti-sense) strand. The codingsequence which encodes the protein of the invention may be identical toone of the coding sequences set forth in the Figures, or may be adifferent coding sequence which as a result of the redundancy ordegeneracy of the genetic code, encodes the same protein as thesequences set forth therein.

A nucleotide sequence which encodes a protein of the present inventionmay include: a coding sequence for the protein or any variant thereof; acoding sequence for the protein or any variant thereof and additionalcoding sequence such as a leader or secretory sequence or a proproteinsequence; a coding sequence for the protein or any variant thereof (andoptionally additional coding sequence) and non-coding sequences, such asintrons or non-coding sequences 5′ and/or 3′ of the coding sequence forthe full length protein.

The invention also provides nucleotide variants, analogues, derivativesand fragments which encode a protein of the invention. Nucleotides areincluded which preferably have at least 65% identity over their entirelength to the nucleotide having the sequence of FIG. 3. More preferredare those sequences which have at least 75% identity over their entirelength to the nucleotide having the sequence of FIG. 3. Even morepreferred are polynucleotides which demonstrate at least 90%, forexample 95%, 97%, 98% or 99% identity over their entire length to thenucleotide having the sequence of FIG. 3.

The nucleotide sequences of the invention may also have the codingsequence fused in frame to one or more marker sequences which allow forpurification of the protein of the present invention such as a FLAGepitope, a myc sequence, or a secretory signal.

The nucleotide sequences of the present invention may be employed forproducing a protein of the invention by recombinant techniques. Thus,for example the nucleotide sequence may be included in any one of avariety of expression vehicles or cloning vehicles, in particularvectors or plasmids for expressing a protein. Such vectors includechromosomal, non-chromosomal and synthetic DNA sequences. Examples ofsuitable vectors include derivatives of bacterial plasmids; phage DNA;yeast plasmids; vectors derived from combinations of plasmids and phageDNA and viral DNA. However, any other plasmid or vector may be used aslong as it is replicable and viable in the host.

More particularly, the present invention also provides a vectorcomprising one or more of the nucleotide sequences as described above.The vectors are, for example, an expression vector, such as a plasmid orviral vector into which an isolated polynucleotide of the invention hasbeen inserted, in a forward or reverse orientation. In a preferredaspect of this embodiment, the vector further comprises one or moreregulatory sequences to direct mRNA synthesis, including, for example, apromoter, operably linked to the sequence. Suitable promoters include:CMV, LTR or SV40 promoter and other promoters known to controlexpression of genes in prokaryotic or eukaryotic cells or their viruses.The vector may contain an enhancer and a ribosome binding site fortranslation initiation and a transcription terminator.

Large numbers of suitable vectors and promoters/enhancers, will be knownto those of skill in the art, but any plasmid or vector,promoter/enhancer may be used as long as it is replicable and functionalin the host.

Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts include mammalian expression vectors, insect expressionvectors, yeast expression vectors, bacterial expression vectors andviral expression vectors and are described in Sambrook et al., MolecularCloning: A laboratory Manual, Second Edition, Cold Spring Harbor, N.Y.,(1989) A preferred vector is pFLAG-CMV-1 or pcDNA3.

The vector may also include appropriate sequences for selection and/oramplification of expression. For this the vector will comprise one ormore phenotypic selectable/amplifiable markers. Such markers are alsowell known to those skilled in the art.

In a further embodiment, the present invention provides host cellscomprising a vector of the invention, and capable of expressing anucleotide sequence of the invention. The host cells can be, forexample, a higher eukaryotic cell, such as a mammalian cell or a lowereukaryotic cell, such as a yeast cell or a prokaryotic cell such as abacterial cell. Suitable prokaryotic hosts for transformation includeE-coli. Suitable eukaryotic hosts include HEK293T cells and HeLa cells.

Cell free translation systems can also be employed to produce suchproteins using RNAs derived from the DNA constructs of the presentinvention.

Routine methods can be employed to purify the protein of the inventionfrom recombinant cell cultures. Such methods are well understood bypersons skilled in the art.

The proteins and nucleotide sequences of the present invention areprovided in an isolated form. The term “isolated” is intended to conveythat the material is not in its native state. Thus, thenaturally-occurring nucleotide sequence or protein present in a livinganimal is in its native state and is not isolated, but the samenucleotide sequence or protein, separated from some or all of thematerials it co-exists with in the natural system, is isolated.Similarly, a protein which has been produced by synthetic means, forexample, by recombinant methods is “isolated.” Such nucleotide sequencecould be part of a vector.

Such nucleotide sequence or protein could be part of a composition, andstill be isolated in that such vector or composition is not part of itsnatural environment. The proteins and nucleotide sequences of thepresent invention are also preferably provided in purified form, andpreferably are purified to at least 50% purity, more preferably about75% purity, most preferably 90% purity or greater, such as 95%, 98%pure.

A further aspect of the present invention is the use of the proteinsaccording to the invention in screening methods. Such methods identifycompounds which act as modulators of the interaction between proteins ofthe invention and their receptor. In general terms, such screens willcomprise contacting a protein of the invention, preferably in trimericform, and its receptor in the presence or absence of the test compound,and measuring the increase or decrease in the level of binding, orincrease or decrease in a response, for example NF-kB activation or CD40activation, when the test compound is present. The proteins of theinvention may be used in high throughput screens, thus enabling largenumbers of compounds to be studied. The screening methods of theinvention are generally well known to persons skilled in the art. Thepresent invention also includes within its scope those compounds whichare identified by the screening methods of the invention as possessinguseful activity.

The present invention further provides compounds which are modulators ofthe interaction between a protein of the invention and its receptor foruse in therapy, for example immunotherapy. The compounds are providedfor use in the treatment of, for example, autoimmune disease,inflammation and other diseases associated with the activation of thetranscription factor NF-κB, for example, rheumatoid arthritis, neuronalinflammation, asthma, in the treatment of cancers, in the treatment ofinfections, such as septic shock and in the treatment ofatherosclerosis. The compounds may be agonists or antagonists of thereceptor to which the proteins of the invention bind, but preferably areantagonists. The compounds include, for example, aptamers, polypeptidesand small molecules.

The invention further provides the use of compounds which have beenidentified by the screening techniques of the invention, for themanufacture of a medicament for use in treatment or prophylaxis ofdisorders that are responsive to modulation of the interaction betweenthe protein of the invention and its receptor.

The present invention additionally provides a method of treatment of adisorder which is responsive to modulation of the interaction betweenthe protein of the invention and its receptor which comprisesadministering to a patient an effective amount of a compoundidentifiable by the screening techniques of the invention, or aneffective amount of the protein of the invention.

The present invention further provides the protein of the invention foruse in therapy, for example, for use in immunotherapy, particularlyduring viral infections, as a vaccine, or as a vaccine adjuvant.

The present invention also provides the use of the protein of theinvention in the manufacture of a medicament for use in immunotherapy,for example, during viral infections, or as a vaccine adjuvant.

The present invention additionally provides a method of treatment of adisorder which is responsive to an increased amount of the protein ofthe invention which comprises administering to a patient an effectiveamount of the protein of the invention.

The invention also provides a nucleotide sequence as defined herein, foruse in gene therapy or as a vaccine, for example, to increase theproduction of the protein of the invention in disorders which respond toan increased level of the protein of the sequence of FIG. 1 or FIG. 2. Apatient may be provided with said nucleotide as a naked polynucleotidein the form of an expression vector such as a plasmid, or with a viralvector comprising said nucleotide, or a cell comprising said nucleotideor vector.

Complementary or anti-sense strands of the nucleotide sequences of theinvention can also be used in gene therapy. For example, a cDNA sequenceor fragments thereof could be used in gene therapy strategies to downregulate expression of the protein of the invention. Antisensetechnology can be used to control gene expression through triple-helixformation of antisense DNA or RNA, both of which methods are based onbinding of a nucleotide sequence to DNA or RNA.

Suitable techniques for introducing the naked polynucleotide or vectorinto a patient include topical application with an appropriate vehicle.The naked polynucleotide or vector may be present together with apharmaceutically acceptable excipient, such as phosphate buffered saline(PBS). One technique involves particle bombardment (which is also knownas ‘gene gun’ technology and is described in U.S. Pat. No. 5,371,015).Here inert particles (such as gold beads) are coated with a nucleicacid, and are accelerated at speeds sufficient to enable them topenetrate a surface of a recipient (e.g. skin), for example by means ofdischarge under high pressure from a projecting device. (Particlescoated with a nucleic acid molecule of the present invention are withinthe scope of the present invention, as are devices loaded with suchparticles.) Other methods of administering the nucleic acid directly toa recipient include ultrasound, electrical stimulation, electroporationand microseeding which is described in U.S. Pat. No. 5,697,901.

Nucleic acid molecules of the present invention may also be administeredby means of specialised delivery vectors useful in gene therapy. Genetherapy approaches are discussed for example by Verme et al, Nature1997, 389:239-242. Both viral and non-viral systems can be used. Viralbased systems include retroviral, lentiviral, adenoviral,adeno-associated viral, herpes viral and vaccinia-viral based systems.Non-viral based systems include direct administration of nucleic acidsand liposome-based systems.

A nucleic acid sequence of the present invention may be administered bymeans of transformed cells. Such cells include cells harvested from asubject. The naked polynucleotide or vector of the present invention canbe introduced into such cells in vitro and the transformed cells canlater be returned to the subject.

The polynucleotide of the invention may integrate into nucleic acidalready present in a cell by homologous recombination events. Atransformed cell may, if desired, be grown up in vitro and one or moreof the resultant cells may be used in the present invention. Cells canbe provided at an appropriate site in a patient by known surgical ormicrosurgical techniques (e.g. grafting, micro-injection, etc.)

The invention also relates to compositions comprising the polypeptide,polynucleotide, vector or transfected cell of the invention in additionto those which may be administered by gene gun. Thus, the polypeptidesof the present invention may be employed in combination with anon-sterile or sterile carrier or carriers for use with cells, tissuesor organisms, such as a pharmaceutical carrier suitable foradministration to a subject. Such compositions comprise, for instance, amedia additive or a therapeutically effective amount of a polypeptide ofthe invention and a pharmaceutically acceptable carrier or excipient.Such carriers may include, but are not limited to, saline, bufferedsaline, dextrose, water, glycerol, ethanol and combinations thereof. Theformulation should suit the mode of administration.

The invention further relates to diagnostic and pharmaceutical packs andkits comprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.Associated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, reflecting approval by theagency of the manufacture, use or sale of the product for humanadministration.

Polypeptides, polynucleotides and other compounds of the presentinvention such as those identifiable by screening methods as describedabove may be employed alone or in conjunction with other compounds, suchas therapeutic compounds. The pharmaceutical compositions may beadministered in any effective, convenient manner including, forinstance, administration by topical, oral, anal, vaginal, intravenous,intraperitoneal, intramuscular, subcutaneous, intranasal or intradermalroutes among others.

The pharmaceutical compositions generally are administered in an amounteffective for treatment or prophylaxis of a specific indication orindications. In general, the compositions are administered in an amountof at least about 10 μg/kg body weight. In most cases they will beadministered in an amount not in excess of about 8 mg/kg body weight perday. Preferably, in most cases, dose is from about 10 μg/kg to about 1mg/kg body weight, daily. It will be appreciated that optimum dosagewill be determined by standard methods for each treatment modality andindication, taking into account the indication, its severity, route ofadministration, complicating conditions and the like.

In therapy or as a prophylactic, the active agent may be administered toan individual as an injectable composition, for example as a sterileaqueous dispersion, preferably isotonic.

Alternatively the composition may be formulated for topical applicationfor example in the form of ointments, creams, lotions, eye ointments,eye drops, ear drops, mouthwash, impregnated dressings and sutures andaerosols, and may contain appropriate conventional additives, including,for example, preservatives, solvents to assist drug penetration, andemollients in ointments and creams. Such topical formulations may alsocontain compatible conventional carriers, for example cream or ointmentbases, and ethanol or oleyl alcohol for lotions. Such carriers mayconstitute from about 1% to about 98% by weight of the formulation; moreusually they will constitute up to about 80% by weight of theformulation.

For administration to mammals, and particularly humans, it is expectedthat the daily dosage level of the active agent will be from 0.01 mg/kgto 10 mg/kg, typically around 1 mg/kg. The physician in any event willdetermine the actual dosage which will be most suitable for anindividual and will vary with the age, weight and response of theparticular individual. The above dosages are exemplary of the averagecase. There can, of course, be individual instances where higher orlower dosage ranges are merited, and such are within the scope of thisinvention.

The present invention further provides a method of producing a proteinof the invention, which method comprises introducing into an appropriatecell line a vector comprising a polynucleotide as defined herein underconditions suitable for obtaining expression of the protein.

The present invention further provides a method of producing trimerscomprising the protein of the invention, which method comprisesintroducing into an appropriate cell line a vector comprising apolynucleotide as defined herein under conditions suitable for obtainingexpression of the protein, and allowing the protein produced to forminto trimers.

As shown in Example 6, the protein of the invention binds to B celllines such as the Burkitt Lymphoma cell line Raji, the B lymphoma cellline ROMI 8866 and PRM18826. As shown in example 7, in preparations ofwhole blood, the protein of the invention binds only to B cells, not Tcells. These examples confirm the presence of the receptor for theprotein of the invention on B cells, and support the role of the proteinof the invention in regulation of the immune system, and diseases asdescribed above. This is also supported by the fact that the protein ofthe invention is strongly expressed in cells and tissues of the immunesystem as shown in example 5.

One of the first events induced by most members of the TNF ligand familyis activation of NF-κB. Mukhopadhyay et al (Journal of BiologicalChemistry Vol. 274 issue 23 Jun. 4, 1999, pp 15978-15981) havedemonstrated that the protein of the invention can activate NF-κB in adose and time dependent manner. The range of doses used was 1 pM to 1000pM. Treatment of cells with as little as 1 pM of the protein of theinvention produced an increase in NF-κB activation compared to untreatedcells. The activation of this important transcription factor suggeststhat the protein of the invention may be involved in activation ofinflammatory pathways, Molecules that modulate the interaction of theprotein of the invention with its receptor will hence be able tomodulate the activation of NF-κB and so will be useful in any diseasesthat are responsive to modulation of the level of activity of NF-κB, forexample diseases of the immune system such as autoimmune disease,inflammatory diseases such as rheumatoid arthritis, neuronalinflammation and asthma and the proliferative diseases such as cancer.Mukhophadhyay et al further demonstrate that activation of NF-κB in thesame system can be inhibited by antibodies specific to the protein ofthe invention. According to Mukhophadhyay et al, the protein of theinvention was incubated with the specific antibodies before being usedto treat cells. In contrast to the previous experiment, reducedactivation of NF-κB was observed.

It is postulated that the presence of the antibody affects the bindingof the protein of the invention to its receptor, thus preventinggeneration of a signal and consequently reducing NF-κB activation. Thesefindings indicate that other compounds, for example small molecules,which modulate the interaction between the protein of the invention andits receptor can be identified in a screen and can be used to modulateNF-κB activation and other downstream effects.

In Example 9, Chromosomal localisation experiments show that the geneencoding the protein of the invention maps to human chromosome 13,region q33. No other TNF ligand family members have been mapped to thisregion. Abnormalities in this locus have been characterised in BurkittLymphomas as the second most frequent defect (Berger et al GenesChromosomes Cancer. 1:115-118). Also, as shown in Example 6, the proteinof the invention binds strongly to the Burkitt lymphoma cell line Raji,and Schneider et al (as referenced above) have demonstrated that thesoluble form of the protein of the invention binds strongly to otherBurkitt Lymphoma cell lines such as BJAB, Namalawa, Ramos and JIYOYE.

Mukhophadhy et al (as referenced above) have demonstrated that theprotein of the invention is able to inhibit the growth of human tumourcell lines. Activation of NF-κB is an early cellular response which isgenerally followed by cytotoxic effects to tumour cells. By treatingvarious cell lines with the protein of the invention and examining themfor viability, the authors were able to show that there is a dosedependent decrease in the viability of cells in the presence of theprotein of the invention. This was demonstrated for a human histiocyticlymphoma cell line, a prostate cancer cell line, a colon cancer cellline, a cervical carcinoma cell line and a breast carcinoma cell line.

These facts suggest that the protein of the invention may play animportant role in the regulation of tumour development, and thatmolecules that can modulate the interaction of the protein of theinvention with its receptor may be useful in the treatment of cancer.Also, the protein of the invention itself, in its membrane bound or itssoluble form, may be useful in the treatment of cancer.

Schneider et al (Journal of Experimental Medicine volume 189 number 11Jun. 7, 1999 pp 1747-1756) have demonstrated that B cell growth can becostimulated by the full length protein of the invention (i.e. themembrane bound form) as well as by the soluble form of the protein ofthe invention. Hence either form can be utilised in therapy, or to formthe basis of a screen for small molecules which can modulate theinteraction between the protein of the invention and its receptor.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows sequence ID No. 1—the amino acid sequence of the solublehuman form of the protein of the invention. Receptor binding sites areshown in bold, and potential N-linked glycosylation sites are markedwith a dot.

FIG. 2 shows sequence ID No. 2—the amino acid sequence of the humanmembrane bound form of the protein of the invention, which compriseswithin it the soluble form of Seq ID No. 1. Annotations are as forFIG. 1. The transmembrane sequence is underlined.

FIG. 3 shows sequence ID No. 3—the cDNA nucleotide sequence whichencodes the amino acid sequence of Seq ID No. 9, aligned to the aminoacid sequence of Seq ID No. 9. Receptor binding regions are boxed, andpotential N-linked glycosylation sites are marked with a dot.

FIG. 4 shows sequence ID No. 4—the cDNA nucleotide sequence whichencodes the amino acid sequence of Seq ID No. 10, aligned with the aminoacid sequence of Seq ID No. 10. Annotations are as for FIG. 3. Thetransmembrane sequence is underlined.

FIG. 5 shows sequence ID No. 5—the amino acid sequence of the solublemouse form of the protein of the invention. The N-linked glycosylationsite is marked with a dot.

FIG. 6 shows sequence ID No. 6—the amino acid sequence of the mousemembrane bound form of the protein of the invention. Annotations are asfor FIG. 5. The transmembrane region is underlined.

FIG. 7 shows sequence ID No. 7—the cDNA nucleotide sequence that encodesthe amino acid sequence of Seq ID No. 5, aligned with the amino acidsequence of Seq ID No. 5. Annotations are as for FIG. 5.

FIG. 8 shows sequence ID No. 8—the cDNA nucleotide sequence that encodesthe amino acid sequence of Seq ID No. 6, aligned with the amino acidsequence of Seq ID No. 6. Annotations are as for FIG. 6.

FIG. 9 shows an alignment between the mouse and human forms of the fulllength form of the protein of the invention. (Seq ID No. 2 and Seq IDNo. 6)

FIG. 10 Shows analysis of induction of CD40 in the presence (shaded) orabsence (unshaded) of the recombinant soluble human form of the proteinof the invention (A) or IL-4 (B).

FIG. 11 shows Northern Blot analysis of the tissue specific expressionof the protein of the invention in normal mouse (A) and human tumourcell lines (B).(B) tissues.

FIG. 12 shows Northern Blot analysis of the tissue specific expressionof the protein of the invention in immune related tissue (A) and humantumour cell lines (B).

FIG. 13 shows cell binding data of FLAG-sD7 (as defined in example 3below). The shaded area indicates binding to the B-cell lymphoma cellline RPMI 8866. In the absence of FLAG-sD7 no binding is seen (dottedline)

FIG. 14 shows binding of the FLAG-sD7 to CD19+ Bcells (A) and CD3+Tcells (B) in whole blood, demonstrating the specificity of binding, toB cells only.

FIG. 15 shows gel filtration of recombinant FLAG-sD7, and the subsequentSDS-PAGE analysis of the fractions shown to contain protein. Theseresults indicate that the soluble form of the protein of the inventionis able to trimerise.

Throughout the examples:

the protein having the amino acid sequence as shown in FIG. 1 will betermed soluble D7 ligand, and the protein having the amino acid sequenceas shown in FIG. 2 will be termed D7 ligand.

EXAMPLE 1 Use of Soluble D7 Ligand in a Screen to Identify Compoundsthat Modulate the Interaction Between the D7 Ligand and its Receptor

All incubations are done at room temperature

Costar RIA/EIA high binding plates are coated with goat anti-human IgG(Sigma I3382) at 2 μg/ml in PBS overnight. The coating antibody isremoved, and the plates are blocked for at least 2 hours in PBS/2% (w/v)BSA. Plates are then washed three times with PBS/0.1% (v/v) Tween20.

100 μl receptor-Fc (1 μg/ml) in PBS/1% (w/v) BSA/0.1% (v/v) Tween20 isadded, and plates are incubated for 1 hour. Plates are washed five timeswith PBS/0.1% (v/v) Tween20.

Biotin-soluble D7 ligand dilutions in PBS/1% BSA/0.1% Tween20 are added,and plates are incubated for 1 hour. Plates are washed five times withPBS/0.1% (v/v) Tween20.

Streptavidin alkaline phosphatase (1:1000) (Amersham RPN1234) is added,and plates are incubated for 1 hour. Plates are washed five times withPBS/0.1% (v/v) Tween20.

Binding is detected using Life Technologies amplifier solutions(19589-019).

EXAMPLE 2 A Cell Based Screen to Identify Compounds that Modulate theInteraction Between the Soluble D7 Ligand and its Receptor

A general protocol for using a cell based screen to identify compoundsthat modulate the interaction between the soluble D7 ligand and itsreceptor is as follows:

A B cell line known to bind and respond to the D7 ligand is treated withrecombinant soluble human D7 ligand exemplified (e.g. FLAG-shD7 asexemplified below) for a defined time.

Cells are harvested, and the response assayed (The response may bepossibly proliferation, apoptosis, NF-κB activation or cytokineproduction). The assay enables determination of whether the addition ofcompounds inhibits the induction of a response in target cells.

A specific example is as follows:

Soluble D7 ligand upregulates CD40

L3055 Burkitt's lymphoma cell line was grown on a feeder layer of humanfoetal fibroblast cells (HFF515) in L3 medium (RPMI 1640+10% SerumSupreme+antibiotics). HEK293 cells were grown in DMEM supplemented with10% foetal calf serum and antibiotics. L3055 cells were treated eitherwith control medium (four parts L3 medium plus one part HEK 293T cellsupernatant) or with sD7 medium (four parts L3 medium plus one part cellsupernatant from HEK293T cells harvested 24 hours after transienttransfection with sD7) or with IL-4 (control medium plus 200 U/mlrecombinant human IL-4 [Sigma]) and incubated at 37° C. for 72 hours.Cells were harvested and washed once in binding buffer then stained withFITC-conjugated mouse anti-human CD40 (Transduction Laboratories) atroom temperature. The cells were then washed twice in binding bufferbefore analysis by flow cytometry. Induction of CD40 was observed ontreatment with sD7 for 72 hours compared to the control. This assay isrepeated in the presence of a molecule which inhibits or increases theinduction of the response in the target cells, and the results compared.Inhibition/increase of response can be clearly demonstrated. The endresult of CD40 upregulation is that B cells are signalled for growth anddifferentiation. Thus, this experiment supports a role for the D7 ligandin the management of immune responses, and in diseases of the immunesystem such as inflammation.

EXAMPLE 3 Synthesis and Purification of the Soluble D7 Ligand

Nucleic acid encoding the soluble human D7 ligand (amino acids 133 to285) was generated by PCR using the cloned full-length open readingframe as a template.

Nucleic acid encoding the soluble human ligand D7 was cloned into vectorpFLAG-CMV-1 (Kodak) (containing a CMV promoter, a preprotrypsin leadersequence, an amino-terminal FLAG epitope and a human growth hormonepolyA addition sequence) to form construct pFLAG-CMV-1-hsD7.

5×10⁶ HEK 293T cells were resuspended in 250 μl cytomix (120 mM KCl;0.15 mM CaCl₂; 10 mM K₂HPO₄/KH₂PO₄, pH 7.6; 25 mM Hepes, pH 7.6; 2 mMEGTA, pH7.6; 5 mM MgCl₂; 2 mM ATP; 5 mM glutathione; pH adjusted withKOH) containing 25 μg pFLAG-CMV-1-hsD7. Transfection was carried out byusing a BioRad gene pulser (960 μF, 270V).

Following transfection, cells were left on ice for 10 min, thentransferred to a 75 cm² tissue culture flask containing 15 ml medium(DMEM, 10% FCS, 2 mM L-glutamine, penicillin (5 μg/ml) and streptomycin(5 μg/ml)). Medium containing secreted ligand was harvested after 48 hand applied to an affinity chromatography column containing anti-FLAG M2antibody coupled to agarose (Kodak). This was washed with Tris-bufferedsaline (pH 7.4) and fractions were eluted in 0.1M citrate buffer (pH2.5). Fractions were immediately neutralised with 0.2 volumes 1MTris.HCl (pH 7.6).

Fractions containing a human soluble D7 ligand linked to the FLAGepitope (FLAG-hsD7) were identified by Western blotting using M2anti-FLAG antibody. These fractions were pooled, and concentrated usinga Centricon Plus-20 (NWML 5000) column (Millipore). FLAG-hsD7 ligand wasstored at −70°

EXAMPLE 4 Synthesis and Purification of the D7 Ligand

The open reading frame of human D7 ligand is cloned into vector pcDNA3(containing a CMV promoter and a bovine growth hormone polyA additionsignal) to form construct pcDNA3-hD7.

Plasmid pcDNA3-hD7 is transiently transfected by electroporation intoHEK 293T cells (protocol as in example 3).

Cells are harvested after 48 h, and homogenised using a Douncehomogeniser in three volumes of protein extraction buffer (25 mM HepespH 7.4, 0.5% Triton-X-100, 1 “complete” protease inhibitor cocktailtablet (Boehringer Mannheim) per 50 ml buffer).

The D7 ligand is purified by affinity chromatography using anti-D7antibody coupled to agarose.

EXAMPLE 5 Northern Blot Analysis of the Tissue Distribution of the D7Ligand

cDNA coding for human D7 ligand was excised from pCDNA3-hD7 (see example4) with the restriction enzymes BamH1 and Xba1. This cDNA fragment waslabelled with ³²P dCTP using the Amersham ready-prime system accordingto the manufacturers protocol. A 5 μl aliquot of this mixture was mixedwith 10 ml Expresshyb solution (Clontech 8015-1) and the resultingmixture was incubated with one of the following clontech blots: Mouse(7762-1), Human-1 (#7760), Human Cancer Cell line (#7757) or HumanImmune System II (#7768-1); for 2 hours at 65° C. with shaking. Theprobe solution was then removed and the blot was washed successivelywith 2×SSC (saline sodium citrate), 0.05% SDS at room temperature forthree 20 minute periods. This was followed with one wash with 0.1% SSC,0.1% SDS at room temperature. The blot was then exposed to Kodak XAR-5film at −70° C. for 48 hours.

The results of the Northern blot analysis show that D7 ligand RNA isexpressed in heart, lung, spleen, kidney and skeletal muscle but notbrain in both mice (FIG. 111A) and humans (FIG. 11B). A blot ofimmune-related tissues demonstrates strong expression of D7 ligand RNAin spleen, lymph node, thymus, appendix, bone marrow and peripheralblood leukocytes (FIG. 12A) supporting its potential role as a regulatorof immune system functions. Analysis of RNA from a range of human tumourcell lines shows expression of D7 ligand RNA in HL-60 promyelocyticleukaemia cells but not in a range of other tumour cell lines (FIG.12B). The presence of D7 ligand in a leukaemic cell line also supportsthe fact that the D7 ligand is involved in immune system regulation anddisorders.

EXAMPLE 6 Detection of Cell Surface Binding of FLAG-sD7

10⁶ cells were incubated with 50 ng FLAG-hsD7 ligand (see example 3) inbinding buffer (PBS/2.5% FCS/0.1% sodium azide) for 10 minutes at roomtemperature. After washing once in binding buffer, cells were incubatedwith 1 μg anti-FLAG M2 antibody for 10 minutes at room temperature.Cells were washed once in binding buffer, then incubated with 150 μgphycoerythrin-conjugated anti-mouse antibody for 10 minutes at roomtemperature. Following two further washes in binding buffer, flowcytometry was performed using a Coulter XL benchtop flow cytometer anddata were collected on 10⁴ viable cells.

Results of one such experiment are shown in FIG. 13. No significantsignal was detectable when any of the lines tested were treated withanti-FLAG M2 antibody and R-phycoerythrin-conjugated second antibodyonly, but after prior treatment with FLAG-sD7, the signal clearlydemonstrates that FLAG-hsD7 binds to the B lymphoma cell line RPMI 8866.Experiments with other cell lines have shown that FLAG-hsD7 binds twoother B cell lines (RPMI 8226 and Raji) but does not bind to the T celllines H9 and Jurkat, or the myelomonocytic lineage lines HL-60, U937 orTHP-1. These results show that the extracellular domain of human D7ligand binds to B cells, supporting its potential role in regulation ofthe immune system, and also suggesting that expression of the D7receptor is restricted to B cells.

EXAMPLE 7 Detection of Cell Surface Binding of Flag-sD7 in Whole Blood

Whole blood from healthy volunteers was diluted 1:10 with 3.8% (w/v)sodium citrate. 100 ul was used in each binding assay. Cell surfacebinding of FLAG-sD7 was detected as in example 6, except that the secondantibody was Alexa 488-conjugated anti-mouse IgG (Molecular Probes) and,after one wash, cells were incubated with 1 μg PE-conjugated mouseanti-human CD3 (Becton Dickinson) or PE-conjugated mouse anti-human CD19(Coulter-Immunotech). This experiment confirms the specificity ofbinding of FLAG-sD7 demonstrated in Example 6. From FIG. 13 it can beseen that although both B cells and T cells were present, FLAG-sD7 boundonly the CD19+ Bcells (FIG. 14A), and not the CD3+Tcells (FIG. 14B).This confirms the specificity of binding to B cells seen with celllines, and again suggests that expression of the D7 receptor isrestricted to B cells.

EXAMPLE 8 FLAG-sD7 is Able to Trimerise

Purified recombinant FLAG-sD7 was fractionated on a Superose 12 column(Pharmacia). Proteins were eluted in PBS and fractions (1 ml) wereanalysed by Western blotting using anti-FLAG M2 antibody (Sigma). Thecolumn was calibrated with standard proteins: apoferritin (443 kDa),b-amylase (200 kDa), ADH (150 kDa), BSA (66 kDa), carbonic anhydrase (29kDa) and cytochrome C (12.5 kDa). FIG. 15A shows the eluate from thecolumn. FIG. 15B shows the fractions containing the peaks seen in FIG.15A run on an SDS-PAGE.

The SDS-PAGE markers are on the left hand side, indicating that thedenatured protein runs at approximately 22 kDa. The markers on the topof the gel are the standard proteins used to calibrate the column, andthey show that FLAG-sD7 elutes in the gel filtration fractionscorresponding to a molecular weight of approximately 70 to 25 kDa. Theseexperiments demonstrate that FLAG-sD7 is able to assemble correctly intoa homotrimer, with molecular weight approximately 3×22 kDa.

EXAMPLE 9 FISH Mapping of the D7 Ligand

Lymphocytes isolated from human blood were cultured in a-minimalessential medium (a-MEM) supplemented with 10% foetal calf serum andphytohemagglutinin at 37° C. for 68-72 hours. The lymphocyte cultureswere treated with BrdU (0.18 mg/ml Sigma) to synchronise the cellpopulation. The synchronised cells were washed three times with serumfree medium to release the block and recultured at 37° C. for 6 hours ina-MEM with thymidine (2.5 μg/ml Sigma). Cells were harvested and slideswere made by using standard procedures including hypotonic treatment,fixation and air-dry.

Slides were baked at 55° C. for 1 hour. After RNase treatment, theslides were denatured in 70% formamide in 2×SSC for 2 min at 70° C.followed by dehydration with ethanol. D7-27 DNA probe which is the fulllength D7 cDNA as shown in FIG. 4, plus 150 nucleotides of 5′untranslated region and 50 nt of 3′ untranslated region) wasbiotinylated with dATP, and probes were denatured at 75° C. for 5 min.in a hybridisation mix consisting of 50% formamide and 10% dextransulphate. Probes were loaded on the denatured chromosomal slides. Afterover night hybridisation, slides were washed and detected as well asamplified. FISH signals and the DAPI banding was recorded separately bytaking photographs, and the assignment of the FISH mapping data withchromosomal bands was achieved by superimposing DISH signals with DAPIbanded chromosomes. The DAPI banding showed that the signal mapped tohuman chromosome 13, and the FISH results further mapped it to regionq33.

The invention claimed is:
 1. A method of inhibiting the interaction in amammal between a receptor on B-cells and a homotrimeric polypeptidecomprising a first polypeptide having 99% sequence identity to SEQ IDNO: 1, said method comprising administering to said mammal an antibodyor a fragment thereof that specifically binds a second polypeptideconsisting of SEQ ID NO:2.
 2. The method of claim 1, wherein theantibody or fragment thereof binds to the extracellular domain of saidsecond polypeptide.
 3. The method of claim 1, wherein said mammal is ahuman.
 4. The method of claim 1, wherein said antibody is a monoclonalantibody.
 5. A method of inhibiting the binding of a receptor on B-cellswith a homotrimeric polypeptide comprising a first polypeptide having99% sequence identity to SEQ ID NO: 1, said method comprising contactingsaid homotrimeric polypeptide with an antibody or fragment thereof thatspecifically binds a second polypeptide consisting of SEQ ID NO:2. 6.The method of claim 5, wherein the antibody or fragment thereof binds tothe extracellular domain of said second polypeptide.
 7. The method ofclaim 5, wherein said antibody is a monoclonal antibody.